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Bisphenol A (BPA) is a ubiquitous industrial chemical that has been identified as an endocrine disrupting compound (EDC). There is growing concern that early life exposures to EDCs, such as BPA, can adversely affect the male reproductive tract and function. This study was conducted as part of the Consortium Linking Academic and Regulatory Insights on BPA Toxicity (CLARITY-BPA) to further delineate the toxicities associated with continuous exposure to BPA from early gestation, and to comprehensively examine the elicited effects on testes and sperm. NCTR Sprague Dawley rat dams were gavaged from gestational day (GD) 6 until parturition, and their pups were directly gavaged daily from postnatal day (PND) 1 to 90 with BPA (2.5, 25, 250, 2500, 25,000, 250,000 μg/kg/d) or vehicle control. At PND 90, the testes and sperm were collected for evaluation. The testes were histologically evaluated for altered germ cell apoptosis, sperm production, and altered spermiation. RNA and DNA isolated from sperm were assessed for elicited changes in global mRNA transcript abundance and altered DNA methylation. Effects of BPA were observed in changes in body, testis and epididymis weights only at the highest administered dose of BPA of 250,000 μg/kg/d. Genome-wide transcriptomic and epigenomic analyses failed to detect robust alterations in sperm mRNA and DNA methylation levels. These data indicate that prolonged exposure starting in utero to BPA over a wide range of levels has little, if any, impact on the testes and sperm molecular profiles of 90 day old rats as assessed by the histopathologic, morphometric, and molecular endpoints evaluated., (Copyright © 2018. Published by Elsevier Inc.)

The human testis is sensitive to toxicant-induced injury but current methods for detecting adverse effects are limited, insensitive and unreliable. Animal studies use sensitive histopathological endpoints to assess toxicity, but require testicular tissue that is not available during human clinical trials. More sensitive and reliable molecular biomarkers of testicular injury are needed to better monitor testicular toxicity in both clinical and preclinical. Adult male Wistar Han rats were exposed for 4weeks to compounds previously associated with testicular injury, including cisplatin (0, 0.2, 0.3, or 0.4mg/kg/day), BI665915 (0, 20, 70, 100mg/kg/d), BI665636 (0, 20, 100mg/kg/d) or BI163538 (0, 70, 150, 300mg/kg/d) to evaluate reproductive toxicity and assess changes in sperm mRNA levels. None of the compounds resulted in any significant changes in body, testis or epididymis weights, nor were there decreases in testicular homogenization resistant spermatid head counts. Histopathological evaluation found that only BI665915 treatment caused any testicular effects, including minor germ cell loss and disorganization of the seminiferous tubule epithelium, and an increase in the number of retained spermatid heads. A custom PCR-array panel was used to assess induced changes in sperm mRNA. BI665915 treatment resulted in a significant increase in clusterin (Clu) levels and decreases in GTPase, IMAP family member 4 (Gimap4), prostaglandin D2 synthase (Ptgds) and transmembrane protein with EGF like and two follistatin like domains 1 (Tmeff1) levels. Correlation analysis between transcript levels and quantitative histopathological endpoints found a modest association between Clu with retained spermatid heads. These results demonstrate that sperm mRNA levels are sensitive molecular indicators of testicular injury that can potentially be translated into a clinical setting., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Protein covalent binding by xenobiotic metabolites has long been associated with target organ toxicity but mechanistic involvement of such binding has not been widely demonstrated. Modern biochemical, molecular, and immunochemical approaches have facilitated identification of specific protein targets of xenobiotic covalent binding. Such studies have revealed that protein covalent binding is not random, but rather selective with respect to the proteins targeted. Selective binding to specific cellular target proteins may better correlate with toxicity than total protein covalent binding. Current research is directed at characterizing and identifying the targeted proteins and clarifying the effect of such binding on their structure, function, and potential roles in target organ toxicity. The approaches employed to detect and identify the tartgeted proteins are described. Metabolites of acetaminophen, halothane, and 2,5-hexanedione form covalently bound adducts to recently identified protein targets. The selective binding may influence homeostatic or other cellular responses which in turn contribute to drug toxicity, hypersensitivity, or autoimmunity.

The Sertoli cell toxicant 2,5-hexanedione (2,5-HD) causes irreversible testicular atrophy in rats. After toxicant exposure, only Sertoli cells, stem cells, and a few spermatogonia remain in the seminiferous epithelium. In this study, the number, type, and fate of the remaining germ cells were determined. Male Sprague-Dawley rats were exposed to 1% 2,5-HD in drinking water for 5 weeks and then sacrificed 12 or 40 weeks after the start of exposure. Cell counts determined that the stem cell population was diminished in size, but made up a significant portion of the remaining germ cells. The remaining germ cells were primarily type A spermatogonia. Modeling of spermatogonial divisions suggested that most spermatogonia undergo degeneration at the level of type A3 spermatogonia after 2,5-HD-induced atrophy. Apoptosis was demonstrated to occur in the remaining germ cells by nuclear morphology and in situ analysis of DNA fragmentation. Quantitation indicated that apoptosis occurred in a majority of stem cell progeny. We conclude that the irreversibility of 2,5-HD-induced testicular injury results from the reduced size of the stem cell population as well as a block in germ cell development at the level of type A spermatogonia.

2,5-hexanedione (2,5-HD) is a Sertoli cell toxicant which causes germ cell loss and testicular atrophy in the rat. The mechanism of germ cell death over the course of 2,5-HD treatment is not known nor is the reason why residual germ cells do not repopulate the seminiferous epithelium following toxicant withdrawal. In the current study, the role of apoptosis in germ cell loss was studied. Male Fischer rats were treated for up to 5 weeks with 1% 2,5-HD in the drinking water and killed between 0 and 12 weeks after the start of toxicant exposure. Apoptosis was assessed in control and treated animals by (1) DNA fragmentation detected by gel electrophoresis, (2) cellular morphology on plastic sections, and (3) DNA fragmentation in situ by terminal deoxy-nucleotidyl transferase-mediated digoxigenin-UTP nick end label (TUNEL) staining of testis cross sections. All three indices demonstrated a substantial increase in apoptosis which peaked at 5 weeks of 2,5-HD treatment. Morphological analysis determined that apoptosis occurred in germ cells of the seminiferous epithelium. DNA fragmentation determined by gel electrophoresis was barely detectable until 5-6 weeks of toxicant exposure. However, TUNEL staining of testis cross sections indicated that germ cell apoptosis increased after as early as 2 weeks of toxicant exposure, providing a highly sensitive biological marker of toxicant-induced testicular injury. These data also suggested a differential sensitivity of germ cells to toxicant exposure with spermatid apoptosis occurring first at 4-5 weeks of treatment followed by apoptosis of spermatocytes and spermatogonia between 6 and 12 weeks. Together, these data demonstrate that apoptosis is the mechanism of germ cell loss in 2,5-HD-induced testicular injury.

Mono-(2-ethylhexyl) phthalate (MEHP) is a widely studied Sertoli cell toxicant. Here we describe alterations in Sertoli cell vimentin filament distribution and the incidence of testicular germ cell apoptosis in young (28-day-old) Fischer rats that were treated with MEHP (2 microgram/kg, po) and killed 0, 3, 6, or 12 hr after exposure. A collapse in vimentin filaments was observed 3 hr after MEHP exposure without accompanying changes in the pattern of Sertoli cell tubulin or actin. A progressive increase in the perinuclear condensation of the vimentin filaments was observed from 6 to 12 hr after exposure. To evaluate the consequences of these Sertoli cell changes on germ cells, the role of apoptosis in MEHP-induced testicular toxicity was examined. DNA isolated from testis of rats 6 and 12 hr after MEHP exposure showed a marked increase in low-molecular-weight DNA resulting from internucleosomal cleavage. In addition, DNA fragmentation visualized in frozen testis cross sections by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick end label (TUNEL) staining demonstrated a progressive increase in germ cell apoptosis from 6 to 12 hr after MEHP exposure. However, 3 hr after MEHP exposure, the incidence of TUNEL-positive germ cells was significantly decreased compared to that seen in controls. Taken together, the early collapse in Sertoli cell vimentin filaments and the concurrent decrease in germ cell apoptosis suggests that MEHP engenders Sertoli cell dysfunction resulting in the disruption of the physiological mechanism of germ cell apoptosis.

Sertoli cells were isolated from 2,5-hexanedione (2,5-HD)-exposed, cryptorchid and 21-day-old rats in order to examine alterations in in vitro Sertoli cell transferrin secretion, germ cell adhesion, in vitro morphology, and cytoskeletal organization which might be involved in the irreversibility of 2,5-HD-induced testicular injury. Sertoli cells isolated from 21-day-old, cryptorchid and 2,5-HD-exposed rats exhibited similar transferrin secretion as measured using an enzyme-linked immunosorbent assay. Germ-cell adhesion was measured using [3H]leucine-labeled immature rat germ cells and revealed similar levels of germ-cell binding in Sertoli cell cultures isolated from the three groups of rats. Differential interference contrast microscopy demonstrated that Sertoli cells isolated from 2,5-HD-exposed rats possessed an atypical spindle shape and long cytoplasmic processes. The immunofluorescent distribution of tubulin and vimentin corresponded with the morphological appearance of the cells with well-defined microtubule and intermediate filament networks which, in the cells isolated from 2,5-HD-exposed rats, extended into the cytoplasmic processes. Rhodamine-conjugated phalloidin-labeled actin stress fibers were decreased in density within the 2,5-HD-exposed rat Sertoli cells. The altered morphology and distribution of actin filaments within Sertoli cells isolated from adult 2,5-HD-exposed rats may reflect an underlying insult which is involved in the irreversible nature of 2,5-HD intoxication.

Cisplatin is an highly effective chemotherapeutic agent which produces a cumulative dose-limiting peripheral neuropathy. In this study, a possible role for microtubule abnormalities in cisplatin-induced toxicity was explored. CD rats (300 g) were injected daily ip with five doses of cisplatin (2 mg/kg) or every other day ip with two doses of cisplatin (10 mg/kg). The day after the last dose, the rats were killed and tubulin was purified from their testes. The maximal rate of cold-induced microtubule disassembly was consistently slower for testis tubulin purified from cisplatin-treated rats compared with control rats. Overnight in vitro coincubation of polymerized bovine brain tubulin with cisplatin followed by a purifying cycle of assembly and disassembly yielded tubulin capable of forming morphologically normal but short microtubules (average length: cisplatin-treated, 2.5 microns; control, 3.7 microns). Cisplatin coincubation markedly reduced the rate of cold-induced microtubule disassembly, producing a half-maximal effect at approximately 0.3 mM. The cold stability of cisplatin-treated microtubules could be partially reversed by diethyldithiocarbamate. Carboplatin, a cisplatin analog which does not cause clinical peripheral neuropathy, was less capable of producing microtubule disassembly abnormalities. These findings demonstrate the ability of cisplatin to alter microtubule disassembly by direct tubulin modification, an abnormality which may contribute to cisplatin-induced peripheral neuropathy.

2,5-Hexanedione (2,5-HD) is a testicular and nervous system toxicant with an unknown mechanism of action. In this study, the effects of 2,5-HD on seminiferous tubule fluid (STF) secretion, testis morphology, and tubulin distribution were examined. Charles River CD rats (200 g) were exposed to 1% 2,5-HD in the drinking water for 5 weeks followed by a 3-week recovery period. STF secretion was measured by efferent duct ligation, and testis cross sections were prepared at 2, 3, 3.43, 3.57, 3.86, 4, and 8 weeks after beginning exposure. A dramatic inhibition of STF secretion was observed between Weeks 3 and 4. The inhibition of STF secretion occurred following simultaneous changes in Sertoli cell and elongate spermatid morphology but prior to changes in round spermatid morphology. Also, alterations in seminiferous tubule tubulin distribution were observed with kinetics similar to those for changes in seminiferous tubule morphology. This temporal sequence suggests a model of 2,5-HD-induced injury in which populations of germ cells are differentially sensitive to impairment of Sertoli cell function.

The effects of 2,5-hexanedione (2,5-HD) exposure on the expression of intermediate filament and actin proteins in adult rat testis was examined during progressive stages of testicular injury. The distribution of vimentin within testis cross sections was examined using immunohistochemistry while rhodamine-labeled phalloidin was used to localize filamentous actin. Keratin was not detected in immunoblots of intermediate filament proteins tested with an antikeratin antibody to investigate the possible reexpression of the prepubertal intermediate filament protein keratin in 2,5-HD-exposed adult testes. However, 2,5-HD exposure did result in a progressively abnormal distribution of actin and vimentin within the seminiferous epithelium with the appearance of a high-molecular-weight protein which was vimentin immunoreactive and not present in control testes.

Zygotes of Lytechinus pictus and Lytechinus variegatus were microinjected with 2,5-hexanedione (2,5-HD)-treated tubulin prior to the first mitotic cycle. Mitotic spindles were small with a well-defined metaphase plate, but poor birefringence and poor astral development. Abnormalities were observed in chromosome movement at anaphase and cytokinesis. Neither microinjections of untreated tubulin or 3-acetyl-2,5-hexanedione-treated tubulin, nor incubation of zygotes in 2,5-HD-containing sea water produced abnormalities. The results can be explained in terms of the nondissociating properties of 2,5-HD-treated tubulin. 2,5-HD-treated tubulin dissociates slowly from microtubules, a property which, besides favoring the formation of stable microtubules, allows this tubulin to induce microtubule assembly when present in substoichiometric amounts. These characteristics have been implicated as a cause of 2,5-HD-induced Sertoli cell dysfunction. The effect of 2,5-HD-treated tubulin on microtubule dynamics in sea urchin zygotes may bear similarities to the effects of 2,5-HD treatment in vivo on Sertoli cell microtubules.

The neurotoxicity of the gamma-diketone, 3,4-dimethyl-2,5-hexanedione, was studied in rats and compared to the known neurotoxicity of the parent compound, 2,5-hexanedione. The test compound was found to be 20 to 30 times more potent on a molar basis than hexanedione. In addition, unlike the distal axonal changes associated with hexanedione, the neurofilamentous swellings following exposure to the dimethyl analog occurred more proximally in the axon, with a preponderance in the anterior horn and lateral tracts of the spinal cord, and in the anterior roots. Since alkyl substitution causes branched-chain compounds to cyclize more rapidly than unbranched analogs, the greater neurotoxicity of the dimethyl compound implicates pyrrole formation in the pathogenesis of n-hexane neuropathy. Furthermore, the location of the axonal swellings induced with 3,4-dimethyl 2,5-hexanedione suggests that there is a common mechanism of injury for the entire class of neurofilament neuropathies, providing a continuum between the intraspinal swellings of beta, beta'-iminodipropionitrile (IDPN) and the distal axonopathies of 2,5-hexanedione, carbon disulfide, and acrylamide. In addition, lower doses of 3,4-dimethyl-2,5-hexanedione for longer periods of time led to a shift in the location of the axonal swellings to include more distal sites. These observations support the hypothesis that covalent crosslinking of the stable neurofilament is the primary event in the molecular pathogenesis of these toxic neuropathies, and that the rate of crosslinking of neurofilaments determines the proximodistal location of the axonal swelling.

Charles River CD rats (200 g) were divided into three groups receiving either 1% 2,5-hexanedione (2,5-HD) or 0.035% 3,4-dimethyl-2,5-hexanedione (DMHD) in the drinking water or water alone (control) for 4 weeks. The two treated groups experienced similar nervous system dysfunction and systemic toxicity. Testicular toxicity, as evidenced by histological changes and decreased testis weight, was present only in 2,5-HD-treated rats. Tubulin was purified from brain and testis and assembly properties were determined. Purified brain and testis tubulin derived from the 2,5-HD-intoxicated rats displayed altered assembly with a shortened nucleation phase and more rapid rate of elongation. Brain tubulin from DMHD-intoxicated rats displayed assembly behavior similar to controls, while testis tubulin from DMHD-intoxicated rats displayed assembly behavior intermediate between the control and 2,5-HD tubulin preparations. The presence of gamma-diketone-induced assembly alterations following in vivo intoxication was accompanied by the formation of a high-molecular-weight protein identified as crosslinked tubulin. From these data, we conclude that microtubule assembly alterations are not etiologic in the development of nervous system dysfunction following intoxication but may represent the biochemical mechanism of 2,5-HD-induced testicular atrophy.

Charles River CD rats (200 g) were intoxicated with 1% 2,5-hexanedione (2,5-HD) in the drinking water for 5 weeks followed by a 17-week recovery period. Pyrrole reactivity of testis proteins increased early during intoxication and then returned toward normal during recovery. Testis tubulin content first increased as germ cells were lost and then fell over time while atrophy was maintained. Purified testis tubulin demonstrated a decreased nucleation time for microtubule assembly at 2 weeks, maintained this alteration throughout intoxication, and then returned to normal assembly kinetics during recovery. The assembly abnormality was accompanied by the presence of a unique crosslinked tubulin species. These findings support the hypothesis that alterations in Sertoli cell microtubules result in germ cell loss following 2,5-HD exposure.

The histopathology of the testicular injury induced by 2,5-hexanedione (2,5-HD) exposure was examined in the rat. Charles River CD rats (200 g) were intoxicated by consuming 1% 2,5-HD in the drinking water or by intraperitoneal injection of the toxicant. Both neurotoxic and subneurotoxic exposures were studied, the total dose ranging from 40 to 211 mmol/kg. The following results were obtained: (1) there was a time delay between administration of the toxicant and development of the testicular injury, (2) Sertoli cell vacuolation in stages associated with the meiotic metaphase was the first histological sign of cellular injury at all doses, (3) subneurotoxic doses produced selective defects in germ cells in stages I-VIII of the spermatogenic cycle, (4) both subneurotoxic and neurotoxic doses produced germ cell necrosis and generalized sloughing of germ cells, and (5) intensive intoxication followed by a 17-week recovery period resulted in an absence of all postspermatogonial germ cells from the seminiferous epithelium of three of five treated rats. These data demonstrate that 2,5-hexanedione-induced testicular atrophy occurs at exposure levels below those producing clinical neurotoxicity and that, within the time frame of this study, the testicular injury is at least partially irreversible.

3,4-Dimethyl-2,5-hexanedione and 2,5-hexanedione were reacted with model amines to yield N-substituted 2,3,4,5-tetramethylpyrroles and 2,5-dimethylpyrroles, respectively. When compared to the unsubstituted parent compound 2,5-hexanedione, 3,4-dimethyl-2,5-hexanedione was found to cyclize approximately eight times as rapidly on a molar basis at 37 degrees C, with an activation energy of 3290 cal/mole less than 2,5-hexanedione. In addition, 1-benzyl-2,3,4,5-tetramethylpyrrole oxidized more readily than 1-benzyl-2,5-dimethylpyrrole with a difference in the half-wave potentials of 0.29 V. Both gamma-diketones led to progressive crosslinking of proteins in vitro, with the dimethyl substitution accelerating this process by a factor of 40. The formation of pyrrolyl derivatives in vivo was demonstrated by the characteristic absorption spectra obtained following reaction of erythrocyte proteins from intoxicated rats with Ehrlich's reagent. There was progressive formation of protein-bound dimethylpyrroles following exposure to 2,5-hexanedione and formation of tetramethylpyrroles following exposure to 3,4-dimethyl-2,5-hexanedione in vivo. Preparations of axonal pads also demonstrated pyrrole derivatization in vivo. In addition, spectrin preparations of erythrocytes from intoxicated rats showed a large amount of high molecular weight protein (400,000 Da), corresponding to dimerized spectrin. Thus, 3,4-dimethyl-2,5-hexanedione, which is 20 to 30 times more potent on a molar basis than 2,5-hexanedione in leading to a neurofilamentous neuropathy, is associated with more rapid pyrrole formation and protein crosslinking in vitro, and it has been demonstrated that these processes occur in vivo. These observations support the hypothesis that pyrrole formation and autoxidation occur following exposure to gamma-diketones, leading to covalent crosslinking of proteins in vivo, a process which may explain the pathogenesis of neurofilament accumulation in these neuropathies.

Numerous studies in recent years have elucidated fundamental properties of axoplasmic structure, biochemistry, and function. The structural role of the cytoskeletal elements, the orientation of MTs within the axon, the phenomenon of MT-dependent transport, and the identity and direction of movement of two MT motors--kinesin and MAP-1C--have been revealed. For many years to come, researchers investigating the structure and function of the Sertoli cell cytoskeleton will be able to adapt techniques gleaned from work on the axonal cytoskeleton. Innovative thinking will be required to apply these techniques to the special circumstances of the male reproductive system; however, the underlying questions are similar. For example, knowledge of several fundamental properties of transport processes in the Sertoli cell would facilitate the toxicologic evaluation of this system. What is the orientation of MTs within the Sertoli cell cytoplasm? Are the fast-growing (+) ends of all MTs in the Sertoli cell cytoplasm directed toward the lumen? This is an important question because the direction of MT-dependent transport involving known MT motors is dependent upon the MT orientation. Which of the Sertoli cell transport pathways are MT-dependent pathways? What are the MT motors involved in these pathways? Ultrastructural examination following exposure to specific cytoskeleton-disrupting agents has highlighted the importance of AFs, IFs, and MTs in the Sertoli cell. Future research will focus on the nature of those molecules which integrate these cytoskeletal components into a dynamic whole, the regulatory systems which control this integration, and the role of an integrated cytoskeleton in Sertoli cell function and testicular homeostasis. Toxicology will be an active participant in this process of scientific discovery. The selective nervous system and testicular toxicants may be useful tools in revealing similarities in the cytoskeletal organization of these apparently disparate organ systems. By searching for common targets in the testis and nervous system, the mechanisms of action of these agents may be more easily, and more confidently, determined.

Charles River CD rats (220 g) were intoxicated with 1.0, 0.5, or 0.25% 2,5-hexanedione (2,5-HD) in the drinking water for a total of 21, 35, or 69 days, respectively. All rats received a total dose of 131 +/- 2 mmol/kg 2,5-HD at dose rates ranging from 1.9 to 6.1 mmol/kg/day. Rats were sacrificed 4 weeks after ending intoxication to evaluate the extent of testicular injury. An exposure rate of 6.1 mmol 2,5-HD/kg/day produced uniformally low testis weights (49% of control) and severe germ cell depletion, while exposure at 1.9 mmol/kg/day gave normal testis weights and histology. Exposure at the intermediate dose rate of 3.8 mmol 2,5-HD/kg/day produced an intermediate degree of testicular injury. In a separate experiment, testis pyrrole content and microtubule assembly behavior were measured in rats exposed to 2,5-HD at the various dose rates for 3 weeks. The rate of intoxication determined the extent of biochemical abnormality. Rats exposed to 1.0, 0.5, or 0.25% 2,5-HD had microtubule nucleation times 55, 63, and 72% of control and pyrrole contents equivalent to 2.14, 1.40, and 1.18 nmol 2,5-dimethylpyrrole/mg testis protein. These data demonstrate that 2,5-HD-induced testicular injury, unlike the nervous system toxicity, is dependent upon the rate of intoxication independent of total dose.

The toxic syndrome resulting from in vivo exposure to n-hexane or n-hexane derivatives may, in part, be a manifestation of altered tubulin and microtubule properties. The effect of in vitro gamma-diketone derivatization was first studied using purified bovine brain tubulin and the results were then verified in tubulins purified from target organs of an experimental species. Microtubule assembly and structure were modified after in vitro incubation with 2,5-hexanedione (2,5-HD) as follows: 2,5-HD derivatization of purified tubulin resulted in an alteration in microtubule assembly kinetics, most prominently a decrease in the length of the nucleation phase, the alteration in assembly kinetics was accompanied by the formation of a covalently crosslinked tubulin dimer, mixing experiments which combined different proportions of control and treated tubulin showed that only a small amount of derivatized tubulin need be present to induce altered assembly properties, and as a result of the more rapid nucleation phase, a greater number of nucleating seeds produced more numerous and shorter assembled polymers. In vitro incubation with the 2,5-HD congener 3,4-dimethyl-2,5-hexanedione produced similar alterations in microtubule assembly. Thus, both the kinetics of tubulin polymerization and the morphology of the final assembly product were modified by in vitro gamma-diketone incubation.